Nedd4-2-dependent regulation of astrocytic Kir4.1 and Connexin43 controls neuronal network activity.

Nedd4-2 is an E3 ubiquitin ligase in which missense mutation is related to familial epilepsy, indicating its critical role in regulating neuronal network activity. However, Nedd4-2 substrates involved in neuronal network function have yet to be identified. Using mouse lines lacking Nedd4-1 and Nedd4-2, we identified astrocytic channel proteins inwardly rectifying K+ channel 4.1 (Kir4.1) and Connexin43 as Nedd4-2 substrates. We found that the expression of Kir4.1 and Connexin43 is increased upon conditional deletion of Nedd4-2 in astrocytes, leading to an elevation of astrocytic membrane ion permeability and gap junction activity, with a consequent reduction of γ-oscillatory neuronal network activity. Interestingly, our biochemical data demonstrate that missense mutations found in familial epileptic patients produce gain-of-function of the Nedd4-2 gene product. Our data reveal a process of coordinated astrocytic ion channel proteostasis that controls astrocyte function and astrocyte-dependent neuronal network activity and elucidate a potential mechanism by which aberrant Nedd4-2 function leads to epilepsy.

L-type Ca2+ channels mediate regulation of glutamate release by subthreshold potential changes.

Subthreshold depolarization enhances neurotransmitter release evoked by action potentials and plays a key role in modulating synaptic transmission by combining analog and digital signals. This process is known to be Ca2+ dependent. However, the underlying mechanism of how small changes in basal Ca2+ caused by subthreshold depolarization can regulate transmitter release triggered by a large increase in local Ca2+ is not well understood. This study aimed to investigate the source and signaling mechanisms of Ca2+ that couple subthreshold depolarization with the enhancement of glutamate release in hippocampal cultures and CA3 pyramidal neurons. Subthreshold depolarization increased presynaptic Ca2+ levels, the frequency of spontaneous release, and the amplitude of evoked release, all of which were abolished by blocking L-type Ca2+ channels. A high concentration of intracellular Ca2+ buffer or blockade of calmodulin abolished depolarization-induced increases in transmitter release. Estimation of the readily releasable pool size using hypertonic sucrose showed depolarization-induced increases in readily releasable pool size, and this increase was abolished by the blockade of calmodulin. Our results provide mechanistic insights into the modulation of transmitter release by subthreshold potential change and highlight the role of L-type Ca2+ channels in coupling subthreshold depolarization to the activation of Ca2+-dependent signaling molecules that regulate transmitter release.

Multiple facets of serotonergic modulation.

The serotonergic system of the central nervous system (CNS) has been implicated in a broad range of physiological functions and behaviors, such as cognition, mood, social interaction, sexual behavior, feeding behavior, sleep-wake cycle and thermoregulation. Serotonin (5-hydroxytryptamine, 5-HT) establishes a plethora of interactions with neurochemical systems in the CNS via its numerous 5-HT receptors and autoreceptors. The facets of this control are multiple if we consider the molecular actors playing a role in the autoregulation of 5-HT neuron activity including the 5-HT1A, 5-HT1B, 5-HT1D, 5-HT2B, 5-HT7 receptors as well as the serotonin transporter. Moreover, extrinsic loops involving other neurotransmitters giving the other 5-HT receptors the possibility to impact 5-HT neuron activity. Grasping the complexity of these interactions is essential for the development of a variety of therapeutic strategies for cognitive defects and mood disorders. Presently we can illustrate the plurality of the mechanisms and only conceive that these 5-HT controls are likely not uniform in terms of regional and neuronal distribution. Our understanding of the specific expression patterns of these receptors on specific circuits and neuronal populations are progressing and will expand our comprehension of the function and interaction of these receptors with other chemical systems. Thus, the development of new approaches profiling the expression of 5-HT receptors and autoreceptors should reveal additional facets of the 5-HT controls of neurochemical systems in the CNS.

Expression of serotonin 1A and 2A receptors in molecular- and projection-defined neurons of the mouse insular cortex.

The serotonin (5-HT) system is the target of multiple anxiolytics, including Buspirone, which is a partial agonist of the serotonin 1A receptor (5-HT1A). Similarly, ligands of the serotonin 2A receptor (5-HT2A) were shown to alter anxiety level. The 5-HT1A and 2A receptors are widely expressed across the brain, but the target region(s) underlying the influence of those receptors on anxiety remain unknown. Interestingly, recent studies in human and non-human primates have shown that the 5-HT1A and 5-HT2A binding potentials within the insular cortex (insula) are correlated to anxiety. As an initial step to define the function of 5-HT transmission in the insula, we quantified the proportion of specific neuronal populations of the insula expressing 5-HT1A or 5-HT2A. We analyzed seven neural populations, including three defined by a molecular marker (putative glutamate, GABA or parvalbumin), and four defined by their projections to different downstream targets. First, we found that more than 70% of putative glutamatergic neurons, and only 30% of GABAergic neurons express the 5-HT1A. Second, within insular projection neurons, 5-HT1A is highly expressed (75-80%) in the populations targeting one sub-nuclei of the amygdala (central or basolateral), or targeting the rostral or caudal sections of the lateral hypothalamus (LH). Similarly, 70% of putative glutamatergic neurons and only 30% of insular GABAergic neurons contain 5-HT2A. Finally, the 5-HT2A is present in a majority of insula-amygdala and insula-LH projection neurons (73-82%). These observations suggest that most glutamatergic neurons can respond to 5-HT through 5-HT1A or 5-HT2A in the insula, and that 5-HT directly affects a limited number of GABAergic neurons. This study defines a molecular and neuroanatomical map of the 5-HT system within the insular cortex, providing ground knowledge to identify the potential role of serotonergic modulation of selective insular populations in anxiety.

Chronic Alcohol Consumption Results in Greater Damage to the Pancreas Than to the Liver in the Rats.

Alcohol consumption increases the risk of type 2 diabetes. However, its effects on prediabetes or early diabetes have not been studied. We investigated endoplasmic reticulum (ER) stress in the pancreas and liver resulting from chronic alcohol consumption in the prediabetes and early stages of diabetes. We separated Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a type-2 diabetic animal model, into two groups based on diabetic stage: prediabetes and early diabetes were defined as occurrence between the ages of 11 to 16 weeks and 17 to 22 weeks, respectively. The experimental group received an ethanol-containing liquid diet for 6 weeks. An intraperitoneal glucose tolerance test was conducted after 16 and 22 weeks for the prediabetic and early diabetes groups, respectively. There were no significant differences in body weight between the control and ethanol groups. Fasting and 120-min glucose levels were lower and higher, respectively, in the ethanol group than in the control group. In prediabetes rats, alcohol induced significant expression of ER stress markers in the pancreas; however, alcohol did not affect the liver. In early diabetes rats, alcohol significantly increased most ER stress-marker levels in both the pancreas and liver. These results indicate that chronic alcohol consumption increased the risk of diabetes in prediabetic and early diabetic OLETF rats; the pancreas was more susceptible to damage than was the liver in the early diabetic stages, and the adaptive and proapoptotic pathway of ER stress may play key roles in the development and progression of diabetes affected by chronic alcohol ingestion.

Heterozygous ambra1 deficiency in mice: a genetic trait with autism-like behavior restricted to the female gender.

Autism-spectrum disorders (ASD) are heterogeneous, highly heritable neurodevelopmental conditions affecting around 0.5% of the population across cultures, with a male/female ratio of approximately 4:1. Phenotypically, ASD are characterized by social interaction and communication deficits, restricted interests, repetitive behaviors, and reduced cognitive flexibility. Identified causes converge at the level of the synapse, ranging from mutation of synaptic genes to quantitative alterations in synaptic protein expression, e.g., through compromised transcriptional or translational control. We wondered whether reduced turnover and degradation of synapses, due to deregulated autophagy, would lead to similar phenotypical consequences. Ambra1, strongly expressed in cortex, hippocampus, and striatum, is a positive regulator of Beclin1, a principal player in autophagosome formation. While homozygosity of the Ambra1 null mutation causes embryonic lethality, heterozygous mice with reduced Ambra1 expression are viable, reproduce normally, and lack any immediately obvious phenotype. Surprisingly, comprehensive behavioral characterization of these mice revealed an autism-like phenotype in Ambra1 (+/-) females only, including compromised communication and social interactions, a tendency of enhanced stereotypies/repetitive behaviors, and impaired cognitive flexibility. Reduced ultrasound communication was found in adults as well as pups, which achieved otherwise normal neurodevelopmental milestones. These features were all absent in male Ambra1 (+/-) mice. As a first hint explaining this gender difference, we found a much stronger reduction of Ambra1 protein in the cortex of Ambra1 (+/-) females compared to males. To conclude, Ambra1 deficiency can induce an autism-like phenotype. The restriction to the female gender of autism-generation by a defined genetic trait is unique thus far and warrants further investigation.

Juvenile manifestation of ultrasound communication deficits in the neuroligin-4 null mutant mouse model of autism.

Neuroligin-4 (Nlgn4) is a member of the neuroligin family of postsynaptic cell adhesion molecules. Loss-of-function mutations of NLGN4 are among the most frequent, known genetic causes of heritable autism. Adult Nlgn4 null mutant (Nlgn4(-/-)) mice are a construct valid model of human autism, with both genders displaying a remarkable autistic phenotype, including deficits in social interaction and communication as well as restricted and repetitive behaviors. In contrast to adults, autism-related abnormalities in neonatal and juvenile Nlgn4(-/-) mice have not been reported yet. The present study has been designed to systematically investigate in male and female Nlgn4(-/-) pups versus wildtype littermates (WT, Nlgn4(+/+)) developmental milestones and stimulus-induced ultrasound vocalization (USV). Neonatal development, followed daily from postnatal days (PND) 4 to 21, including physical development, neurological reflexes and neuromotor coordination, did not yield any differences between Nlgn4(-/-) and their WT littermates. USV in pups (PND8-9) in response to brief separation from their mothers revealed remarkable gender effects, and a genotype influence in females regarding latency to first call. In juveniles (PND22-23), USV monitoring upon exposure to an anesthetized female intruder mouse uncovered a clear genotype effect with reduced USV in Nlgn4(-/-) mice, and again a more prominent phenotype in females. Together, these data support an early manifestation of communication deficits in Nlgn4(-/-) mice that appear more pronounced in immature females with their overall stronger USV as compared to males.

Chronic alcohol consumption, type 2 diabetes mellitus, insulin-like growth factor-I (IGF-I), and growth hormone (GH) in ethanol-treated diabetic rats.

AIMS: Alcohol has deleterious influences on glucose metabolism which may contribute to the development of type 2 diabetes mellitus (T2DM). Insulin-like growth factor I (IGF-I) and growth hormone (GH), which interact with insulin to modulate metabolic control, have been shown to be related to impaired glucose tolerance. This study was conducted to assess the possibility that altered circulating IGF-I and GH levels contribute to the exacerbation of T2DM by alcohol use in type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats and non-diabetic Long-Evans Tokushima Otsuka (LETO) rats. MAIN METHOD: OLETF rats were pair-fed a Lieber-DeCarli Regular Ethanol diet and LETO rats were pair-fed a control diet for 6 weeks. At 6 weeks, an Intraperitoneal Glucose Tolerance Test (IP-GTT) was performed and IGF-I and GH levels were evaluated. KEY FINDINGS: Prior to an IP-GTT, OLETF-Ethanol (O-E) group had significantly a decrease in the mean glucose levels compared to OLETF-Control (O-C) group. At 120 min post IP-GTT, the O-E group had significantly an increase in the mean glucose levels compared to O-C group. The serum IGF-I levels were significantly lower and the serum GH levels were significantly higher in the O-E group than in L-C group. SIGNIFICANCE: These results suggest that IGF-I and GH are prominent in defining the risk and development of T2DM, and may be adversely affected by heavy alcohol use, possibly mediating its diabetogenic effects. Thus, the overall glucose intolerance in the setting of alcoholism may be attributable to inappropriate alteration of IGF-I and GH levels.

Genetic markers of a Munc13 protein family member, BAIAP3, are gender specifically associated with anxiety and benzodiazepine abuse in mice and humans.

Anxiety disorders and substance abuse, including benzodiazepine use disorder, frequently occur together. Unfortunately, treatment of anxiety disorders still includes benzodiazepines, and patients with an existing comorbid benzodiazepine use disorder or a genetic susceptibility for benzodiazepine use disorder may be at risk of adverse treatment outcomes. The identification of genetic predictors for anxiety disorders, and especially for benzodiazepine use disorder, could aid the selection of the best treatment option and improve clinical outcomes. The brain-specific angiogenesis inhibitor I-associated protein 3 (Baiap3) is a member of the mammalian uncoordinated 13 (Munc13) protein family of synaptic regulators of neurotransmitter exocytosis, with a striking expression pattern in amygdalae, hypothalamus and periaqueductal gray. Deletion of Baiap3 in mice leads to enhanced seizure propensity and increased anxiety, with the latter being more pronounced in female than in male animals. We hypothesized that genetic variation in human BAIAP3 may also be associated with anxiety. By using a phenotype-based genetic association study, we identified two human BAIAP3 single-nucleotide polymorphism risk genotypes (AA for rs2235632, TT for rs1132358) that show a significant association with anxiety in women and, surprisingly, with benzodiazepine abuse in men. Returning to mice, we found that male, but not female, Baiap3 knockout (KO) mice develop tolerance to diazepam more quickly than control animals. Analysis of cultured Baiap3 KO hypothalamus slices revealed an increase in basal network activity and an altered response to diazepam withdrawal. Thus, Baiap3/BAIAP3 is gender specifically associated with anxiety and benzodiazepine use disorder, and the analysis of Baiap3/BAIAP3-related functions may help elucidate mechanisms underlying the development of both disorders.

Ex vivo detection for chronic ethanol consumption-induced neurochemical changes in rats.

The aim of this study was to quantitatively investigate the chronic ethanol-induced cerebral metabolic changes in various regions of the rat brain, using the proton high resolution magic angle spinning spectroscopy technique. The rats were divided into two groups (control group: N=11, ethanol-treated group: N=11) and fed with the liquid diets for 10 weeks. In each week, the mean intake volumes of liquid diet were measured. The brain tissues, including cerebellum (Cere), frontal cortex (FC), hippocampus (Hip), occipital cortex (OC) and thalamus (Thal), were harvested immediately after the end of experiments. The ex vivo proton spectra for the five brain regions were acquired with the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence at 500-MHz NMR spectrometer. All of the spectra were processed using the LCModel software, with simulated basis-set file, and the metabolite levels were referenced to total creatine. In the ethanol liquid diet group, there were significant increases in the metabolites ratio levels, as compared to control (Cere: alanine, glutathione, and N-acetlyaspartate; FC: phosphocholine and taurine; Hip: alanine, glutamine, and N-acetylaspartate; OC: glutamine; Thal: alanine, γ-aminobutyric acid, glutamate, glycerophosphocholine, phosphocholine, taurine, and free choline). However, in the ethanol liquid diet group, the myo-inositol levels of the OC were significantly lower. The present study demonstrates how chronic ethanol consumption affects cerebral metabolites in the chronic ethanol-treated rat. Therefore, this result could be useful to pursue clinical applications for quantitative diagnosis in human alcoholism.

Impairment of long-term potentiation in the hippocampus of alcohol-treated OLETF rats.

Type 2 diabetes and chronic heavy alcohol consumption each have been known to be associated with the impairment of hippocampus-dependent cognitive functions. Although both conditions often coexist clinically and there is accumulated evidence of a relationship between the two, the combined effect on hippocampal long-term potentiation (LTP) has not yet been investigated. We compared the effect of type 2 diabetes itself with that of type 2 diabetes with chronic heavy alcohol consumption on the hippocampal LTP using Otsuka Long-Evans Tokushima Fatty (OLETF) rat model, which resembles the characteristics of human type 2 diabetes. Ten of 16-week-old male OLETF rats were randomized into two treatment groups according to weight: the OLETF-Alcohol (O-A, n=5) and the OLETF-Control (O-C, n=5). The rats in the O-A group were fed Lieber-DeCarli Regular EtOH over a 10-week period and the amount of alcohol consumption was 8.42±2.52g/kg/day. To ensure the effect of poor glycemic control on LTP, intraperitoneal glucose tolerance test was performed after a 10-week treatment. The hippocampal LTP was measured by extracellular field excitatory post-synaptic potentials at Shaffer collateral (SC) synapses in the CA1 region. Although the O-A group showed significantly lower fasting and postprandial glucose (P<0.01 and P=0.02, respectively), the hippocampal LTP was more significantly attenuated in the O-A group than the O-C group (P=0.032). The results of this study suggested that chronic heavy alcohol consumption could potentiate the impairment of hippocampal LTP in individuals with impaired glucose tolerance or early type 2 diabetes, even though it did not aggravate, but did improve glycemic control. Clinical attention to chronic heavy drinking will be required in preventing cognitive impairment in individuals with type 2 diabetes.

The change of plasma ghrelin and leptin levels by the development of type 2 diabetes mellitus in patients with alcohol dependence.

BACKGROUND: There have been lots of studies about the relationship between chronic use of alcohol and the development of type 2 diabetes mellitus (T2DM). Chronic use of alcohol can be affected by the altered level of ghrelin and leptin which regulate food-seeking behavior having similar mechanism of controlling alcohol-craving behavior. Those peptides are known to be correlated with T2DM. Ghrelin and leptin also have been regarded as possible regulators of glucose metabolism and insulin function. Hence, there is the possibility that ghrelin and leptin can be related with deteriorated pathophysiology of T2DM in alcoholic patients. METHODS: Patients with alcohol dependence diagnosed by Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision (DSM-IV-TR) underwent an 75 g oral glucose-tolerance test (OGTT), to classify them to normal glucose tolerance (NGT, n = 52), pre-diabetes including impaired glucose tolerance (IGT), impaired fasting glucose level (IFG) and combination of IGT and IFG (Pre-DM, n = 26) and T2DM (n = 24) groups. Fasting plasma ghrelin and leptin levels were compared among groups. RESULTS: There was no difference of ghrelin concentration among the groups but the leptin concentration was significantly different between NGT and T2DM group (p < 0.05). Increased leptin levels were significantly correlated with body mass index (BMI), insulin level, and insulin resistance. CONCLUSIONS: Chronic alcohol drinking might produce leptin resistance which makes leptin significantly correlated with fasting insulin concentration and insulin resistance. Therefore, we suppose that increased level of leptin by chronic alcohol use could be one of the main mechanisms that develop insulin resistance in alcoholic patients.

Chronic ethanol ingestion, type 2 diabetes mellitus, and brain-derived neurotrophic factor (BDNF) in rats.

Chronic alcohol consumption contributes to the development of type 2 diabetes mellitus (T2DM) while decreasing the level of brain-derived neurotrophic factor (BDNF). BDNF may be an important regulator of glucose metabolism, so it may be associated with an increased risk for T2DM in alcoholism. We evaluated the association of chronic heavy alcohol exposure, T2DM and BDNF level. Ten week-old type 2 diabetic OLETF rats and non-diabetic LETO rats of similar weight were used. The rats were randomized by weight into four treatment groups: (1) OLETF-Ethanol (O-E, n=13), (2) OLETF-Control (O-C, n=15), (3) LETO-Ethanol (L-E, n=11), and (4) LETO-Control (L-C, n=14). The ethanol groups were fed an isocaloric liquid diet containing ethanol while the control groups were fed with the same diet containing maltose-dextran over a 6-week period using a pair-feeding control model in order to regulate different caloric ingestion. After 6 weeks of feeding, an Intraperitoneal Glucose Tolerance Test (IP-GTT) was performed and BDNF levels were analyzed. Prior to IP-GTT, the mean glucose levels in the O-E, O-C, L-E, and L-C groups were 90.38±12.84, 102.13±5.04, 95.18±6.43, and 102.36±4.43mg/dL, respectively. Thirty minutes after intraperitoneal injection, the mean glucose levels were 262.62±63.77, 229.07±51.30, 163.45±26.63, and 156.64±34.42mg/dL, respectively; the increased amount of the mean glucose level in the O-E group was significantly higher than that in the O-C group (p<0.05). One hundred twenty minutes after intraperitoneal injection, the mean glucose levels were 167.38±45.37, 121.20±18.54, 106.73±6.94, and 104.57±9.49mg/dL, respectively; the increased amount of the mean glucose level in the O-E group was significantly higher than that in the O-C group (p<0.01). The difference in mean glucose levels between the O-E group and O-C group was still significant even after adjusting for time (p<0.05). Mean BDNF levels were 405.95±326.16, 618.23±462.15, 749.18±599.93, and 1172.00±839.17pg/mL, respectively; mean BDNF level in the O-E group was significantly lower than the L-C group (p<0.05). In conclusion, the results of the present study suggest that chronic heavy alcohol ingestion may aggravate T2DM and may possibly lower BDNF level.